PROJECT SUMMARY/ABSTRACT Bone marrow suppression is a common adverse effect of long-term antibiotic administration, which can in turn leave patients at substantial risk for future infections. Depletion of commensal intestinal bacteria has recently been uncovered as the proximal cause of antibiotic-mediated bone marrow suppression, implicating the microbiome in maintenance of normal hematopoiesis. Commensal bacteria, acting via type I interferon and STAT1, a major transcription factor downstream of interferon signaling, are necessary to promote the normal function of hematopoietic progenitors in the bone marrow. However, because commensal bacteria in the gut are stimulating effects in the distal compartment of the bone marrow, the cell type(s) mediating these responses are unknown. Further, the sufficiency of type I interferon signaling to maintain hematopoiesis, or which commensal bacterial signaling pathways interact with this pathway to drive hematopoiesis, are unknown. Using a murine model of antibiotic-mediated bone marrow suppression to explore these critical questions, this proposal aims to interrogate the pathways and mechanisms underlying the microbiome?s effects on hematopoiesis. Studies will identify in which tissue and cell type(s) type I interferon and STAT1 signaling are required to promote hematopoiesis, specifically by analyzing STAT1 phosphorylation in different tissues, generating bone marrow chimeras, and testing conditional knock-out mice. The sufficiency of type I interferon signaling to maintain hematopoiesis will be determined by characterizing the potential of recombinant interferons or interferon-stimulatory bacterial products to rescue hematopoiesis in antibiotic-treated mice. Finally, this proposal will interrogate interactions between STAT1 and signaling through NOD1, a bacterial product receptor, because both have been implicated in microbiome-mediated hematopoietic regulation. Experiments will define whether these immune factors act in the same pathway, and identify novel factors linking the microbiota with cytokines and metabolites in the bone marrow niche. These rigorous studies build upon both published and preliminary data to clarify the mechanisms underlying the regulation of hematopoiesis by the commensal microbiome. Successful completion of these aims will serve as a critical basis for future studies to develop preventive and therapeutic approaches to combat antibiotic-associated bone marrow suppression. This work is a close collaboration between Dr. Megan Baldridge, expert in the effects on the commensal microbiome on the innate immune system, at Washington University School of Medicine and Dr. Katherine King, expert in immunologic regulation of primitive hematopoiesis, at Baylor College of Medicine, and leverages these complementary areas of expertise to explore the novel field of microbiome-mediated hematopoietic regulation.